Pig red blood cell hexokinase: Evidence for the presence of hexokinase types II and III, and their purification and characterization

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 226, No. 1, October 1, pp. 365-3’76. 1933

Pig Red Blood Cell Hexokinase: Evidence for the Presence of Hexokinase Types II and Ill, and Their Purification and Characterization’

VILBERTO STOCCHI, MAURO MAGNANI, GIUSEPPE NOVELLI, MARINA DACHA, AND GIORGIO FORNAINI’

Istituto di Chimica Biologica, Universitci degli Studi di Urbino, via Sa&, 2410.29 U&no, Italy

Received February 14, 1983, and in revised form May 17, 1933

Pig erythrocytes, in contrast to red blood cells from other mammals (M. Magnani, V. Stocchi, F. Canestrari, M. Dacha, and G. Fornaini (1982) Biochewa. ht. 4,6’73), have been shown to contain hexokinase (EC 2.7.1.1) types II and III. Hexokinase type III is the predominant form, accounts for 98% of the total glucose phosphorylating activity, and has been purified 290,000-fold by a combination of ion-exchange chromatography and affinity chromatography on Sepharose-N-hexanoylglucosamine. The enzyme was shown to be homogeneous by polyacrylamide and sodium dodecyl sulfate-gel electrophoresis. The highest specific activity obtained was 190 units/mg protein with a yield of 60%. Because the amount of hexokinase II was small, it was only partially purified by ion-exchange chromatography. The native proteins have the same molecular weight of 100,000 by gel filtration on Ultrogel AcA44. The apparent isoelectric point of hexokinase type II was shown to be 4.8 and 4.9 pH units, whereas hexokinase type III was shown to have a pI of 4.3 to 4.4 pH units by isoelectric focusing. Both hexokinases are able to phosphorylate several hexoses. However, while hexokinase II shows an apparent Km for glucose of 1.5. 1O-4 M with negative cooperativity (nn = 0.4), hexokinase III shows an apparent Km for glucose of 1.5. 10m5 M and a positive cooperative effect (nn = 1.5). Furthermore, glucose at concentrations higher than 0.4 mM becomes an inhibitor of hexokinase III. Amino acid analysis of hexokinase type III revealed a low number of the aromatic residues Phe, Tyr, and Trp; this is in agreement with the low extinction coefficient of EL&,, = 12.5.

Hexokinase in mammalian tissues exists as four isoenzymes with distinct kinetic properties and tissue distribution (1, 2). These isozymes are also readily distin- guished by their Km values for glucose (3) which are lop5 M for hexokinase I, lop4 M

for hexokinase II, 10e5 M for type III, and lo-’ M for glucokinase (type IV), while, with the exception of the latter, they show the same molecular weight. We have previously reported (4-7) that mammalian red

’ This work was supported by CNR, Minister0 della Pubblica Istruzione and Cassa di Risparmio di Pesaro.

’ To whom correspondence should be addressed.

blood cells contain mainly hexokinase type I or its subtypes. In the course of our studies we also showed that pig erythrocytes contain two different glucose-phosphorylating activities, which differ in glucose affinity and electrophoretic mobility (8). More recently, Dixon and Wilson (9) have obtained similar results.

In this paper we present evidence that these two glucose-phosphorylating activities correspond to hexokinase type II and type III (in the nomenclature of Katzen and Schimke (10)). We also describe the procedure for the purification to homo- geneity of hexokinase type III, which, in pig erythrocytes, represents 98% of the to-

365 0003-9861/83 $3.00 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

STOCCHI ET AL.

tal hexokinase activity. The kinetic and molecular properties of the two hexokinases are also examined. In an accompanying article (ll), some regulatory char- acteristics of the homogeneous enzyme are reported, and a probable physiological role of hexokinase type III in pig erythrocytes is proposed.

EXPERIMENTAL PROCEDURES

~uterials. Coenzymes, enzymes, substrates, and dithiothreitol were obtained from Sigma Chemical Company (St. Louis, MO.) and Boehringer-Mannheim (Mannheim, FRG). Activated CH-Sepharose 4B, DEAE-Sephadex A-50, and Blue Dextran 2000 were purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). DE-52 was from Whatman (Maidstone, UK). Ultrogel AcA44 and Ampholine (pH ranges: 3.5 to 10, 4 to 6, and 5 to 8) were from LKB (Stockholm, Sweden). Hydrochloric acid, sequanal grade, was from Pierce Chemical Company (Rockford, Ill.). All other reagents were of an analytical grade.

Hexokinase assay. Hexokinase activity was measured at 37°C spectrophotometrically in a system coupled with glucose+phosphate dehydrogenase (EC 1.1.1.49) from yeast, Boehringer grade I. The assay mixture contained, in a total volume of 1 ml, 0.135 M glycylglycine (pH 8.1), 5 mg MgATP, 0.5 mrd NADP, 5 mM MgClz, and 0.1 IU of glucose-g-phosphate dehydrogenase. Hexokinase type III was assayed in the presence of 0.25 mM glucose, and hexokinase type II was assayed in the presence of 100 mM glucose. In the latter case the blank obtained without MgATP was routinely subtracted. Initial-rate measurements were performed by following the reduction of NADP+ at 340 nm with a Beckman Model 25 spectrophotom- eter. For each molecule of glucose utilized a molecule of NADP+ was reduced (with the exception of hemolysate where 2 molecules of NADP+ were reduced for each molecule of glucose utilized due to the en- dogenous 6-phosphogluconate dehydrogenase (EC 1.1.1.44)). One unit of hexokinase activity is defined as the amount of enzyme that catalyzes the formation of 1 pmol of glucose 6-phosphate/min at 37°C.

In the sugar-specificity studies the initial rates of ADP production were measured in a coupled-enzyme system with pyruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.1.28). The assay mixture contained, in total volume of 1 ml, 0.25 M Tris-HCl (pH 8.1), 1.5 mM EDTA, 5.0 mM MgClz, 5.0 mrd ATP-MgClz, 0.75 mM phosphoenolpyruvate, 0.2 mM NADH, 1 IU of pyruvate kinase, 1 IU of lactate dehydrogenase, and sugar substrate. One unit of hexokinase activity is defined as the amount of enzyme that catalyzes the production of 1 rmol of ADP/min at 37°C.

Protein estimatim In the hemolysate, hemoglobin

concentration was determined spectrophotometrically at 540 nm with Drabkin’s solution as described by Beutler (12). During the purification procedure, protein was determined according to Lowry et al (13), with bovine serum albumin as a standard, or spectrophotometrically at 280 nm.

Sepharose-N-arni~~nw~l~~arnine. Sephar- ose-N-aminohexanoyl glucosamine was prepared as before (4). The product was stored at +4”C in 5 mM sodium-potassium phosphate buffer, pH 7.5, containing 3 mM KF and 3 mM X-mercaptoethanol.

Sodium dod.ecyl su&ate-pol~amyhmide slab gel elm trophoresis. Electrophoresis was carried out in 7.0% polyacrylamide slab gels containing 0.1% sodium dodecyl sulfate according to Laemli (14) with slight modifications. Protein bands were visualized with 0.2% Coomassie brilliant blue R-250 (from Sigma). Molec- ular weight calibration curves were obtained using fl-galactosidase (M, = 130,000), phosphorylase a (A& = lOO,OOO), bovine serum albumin (A& = 66,000), egg albumin (Af, = 45,00), pepsin (itf, = 34,700), and tripsinogen, PMSl@ treated (A& = 24,000) as standards (15).

Molecular weight estimation The molecular weight of native hexokinase was determined by Ultrogel AcA44 gel filtration using a calibrated column (2.1 X 56 cm). The column was equilibrated with 5 mM sodium-potassium phosphate buffer, pH 7.5, containing 3 mM 2-mercaptoethanol, 3 mM KF, 5 rnM glucose, 0.5 M NaCl, and 9% (v/v) glycerol at 4°C. One-milliliter samples were layered on the surface of the gel, the flow rate was adjusted to 15 to 20 ml/h, and l.O-ml fractions were collected. In order to detect the elution volumes of hexokinase and the molecular weight standards, each fraction was assayed for enzyme activities. The void volume (V,) was determined with Blue Dextran 2060.

Isoelectric f&using. Isoelectric focusing of hexokinases was carried out on LKB 8100 electrofocusing equipment in a glycerol gradient solution and in a pH gradient of 3.5 to 10 and 4 to 8 at 1% Ampholine concentration according to the instructions of the manufacturer. The sample was layered after the pH gradient was formed. The voltage was 600 V and the focusing was completed after 16 h from the intro- duction of the sample. The cooling temperature was 4°C the elution flow rate was 30 ml/h, and fractions of 1 ml were collected and measured for the hexokinase activity and the pH value.

Amino acid analysis. Protein samples were hydro- lyzed in 6 M constant-boiling HCl in sealed, evacuated tubes for 24 h. Hydrolysates were analyzed on a LKB 4400 amino acid analyzer. Tryptophan was estimated by the spectrofluorometric method of Bliss (16).

’ Abbreviations used: PMSF, phenylmethylsulfonyl fluoride.

PIG RED BLOOD CELL HEXOKINASE 367

Preparation of pig liver hexokinme The preparation of soluble pig liver hexokinase extract was performed using a procedure based on that described by Gross- bard and Schimke (17). Fresh liver was minced and then homogenized with 3 vol of 3 rnr.f sodium-potassium phosphate buffer, pH 7.5, containing 5 mM glucose, 1 mM dithiothreitol, and 10% (v/v) glycerol. The homogenate was centrifuged at 105,OOOg for 1 h at 4°C. At this stage, the supernatant fluid contains 1.1 units/ml solubilized hexokinase activity.

RESULTS

Evidence for the Existence in Pig Erythrocytes of Two Distinct Forms of Hexokinase

Two hexokinases were separated by ion- exchange chromatography from pig erythrocyte hemolysates, prepared as described below (Fig. 1). Compared to the isozyme profile found in pig liver these two glucose- phosphorylating activities show the same mobility as hexokinase type II and type III, also confirmed by cochromatography of pig liver homogenate and pig erythrocyte hexokinases.

As we found in the livers of humans (6) and rabbits (7), pig liver, contrary to that previously reported (18), contains all the four hexokinase isozymes. Kinetic analysis (not shown) reveals that these two glucose- phosphorylating activities in pig erythrocytes correspond to hexokinase types II and III.

In fact, it is well known (18) that hexokinase III is inhibited by high concentrations of glucose and hexokinase II has higher K,,, value for glucose than hexokinase type I and type III, exactly as we found for the two pig red cell enzymes. The pig erythrocyte hexokinase isozymic pattern is influenced by the animal’s age. In fact, in adult pig red cells hexokinase type III accounts for 98-99% of the total glucose- phosphorylating activity, whereas newborn pig erythrocytes contain a small amount of hexokinase III and hexokinase type II is the predominant glucose-phosphorylating activity (Figs. 1B and C). A possible physiological significance of this behav- ior is discussed in the accompanying paper (11).

Putijcation of Pig Erythrocyte Hexokinase Type III

All operations were performed at 4°C. All buffers contained 3 mM 2-mercaptoethanol and 3 mM KF.

Step 1: Preparation of the hemolysate. Pig red blood cells were collected, using EDTA as an anticoagulant, from adult animals. The red cells were washed twice with iso- tonic sodium chloride. The buffy coat was removed by suction. After an equal volume of 0.4% (w/v) saponin solution was added, the washed cells were hemolyzed for 2 h. The red cell stroma were then removed by centrifuging the lysate at 13,000g for 1 h.

Step 2: Batch treatment with DEAE-Se- phadex A-50. The hemolysate was mixed with 2 vol of DEAE-Sephadex A-50 suspension, equilibrated in 3 mM sodium-potassium phosphate buffer, pH 7.3, containing 5 mM glucose, and mechanically stirred for 60 min. The suspension was rinsed in a Biichner funnel with the same buffer until the eluate was colorless. This procedure removed the bulk of the hemoglobin while the hexokinase remained bound. The enzyme was eluted with 0.5 M KC1 in phosphate buffer. For a complete hexokinase recovery, the elution procedure was re- peated twice.

Step 3: First ammonium sulfate fractionation. Solid ammonium sulfate was slowly added to the stirred enzyme solution from Step 2 to achieve 30% saturation. The suspension was gently stirred for 30 min and then centrifuged at 16,000g for 30 min. The supernatant solution was removed and brought to 45% saturation with ammonium sulfate. After 30 min, the suspension was centrifuged as before. The 30 to 45% ammonium sulfate precipitate was dissolved in 15 to 20 ml of 5 mM sodium-potassium phosphate buffer, pH 7.5, containing 0.25 mM glucose, and then dialyzed overnight against 500 vol of 5 mM sodium-potassium phosphate buffer, pH 7.5, containing 0.25 mM glucose and 10% (v/v) glycerol. The dialyzed enzyme solution was then centrifuged at 16,OOOg for 20 min.

Step 4: DEAE-cellulose chromatography. The enzyme solution from Step 3 was applied to a column (1.8 X 25 cm) of DEAE-

368 STOCCHI ET AL.

z 5 F 0 4 i 2 -0.1

z

E D : 1500- P

1250 -

1200 -

750 -

500 - , - 0.2

- 0.1

40- P E

30-

20-

- 0.2

- 0.1

20 40 50 80 100 120 140 150

FRACTION NUMBER

FIG. 1. DEAE-cellulose chromatography of hexokinase activities from pig liver (A), adult pig erythrocytes (B), newborn pig red blood cells (C), ammonium sulfate fraction 30 to 45% (D), and ammonium sulfate fraction 60 to 30% (E). 2.5 ml of pig liver homogenate, 2 ml of hemolysate from adult erythrocyte, 2 ml of hemolysate from newborn red cells, 2 ml of ammonium sulfate fraction 30 to 45%, and 12 ml of ammonium sulfate fraction 60 to 80%. prepared as described in the text, were chromatographed on DE-52 columns (0.7 X 20 cm) equilibrated in 5 mM sodium-potassium phosphate buffer, pH 7.5, containing 0.25 rnhd glucose and 5 mM dithiothreitol. The columns were developed with 280 ml of a linear gradient of KC1 from 0 to 0.4 M in the same sodium-potassium phosphate buffer, and operated at 15 to 20 ml/h. Fractions of 1.6 ml were collected.


FRACTION NUMBER

FIG. 2. DEAE-cellulose column chromatography of hexokinase type III. The sample, 20 ml of the 30 to 45% ammonium sulfate fraction, was applied to the column (1.8 X 25 cm) in 5 mrd sodium- potassium phosphate buffer, pH 7.5, containing 10% (v/v) glycerol, 0.25 mM glucose, and 1 mM dithiothreitol, and operated at 70-30 ml/h. The hexokinase activity was eluted with a linear gradient (300 ml in each chamber) of KC1 from 0 to 0.5 M in the same sodium-potassium phosphate buffer, pH 7.5, and 4.5-ml fractions were collected. 0, absorbance at 230 nm; -, KC1 gradient; 0, hexokinase activity.

cellulose (Whatman DE-52) equilibrated with 5 mM sodium-potassium phosphate, pH 7.5, containing 0.25 mM glucose, 1 mM dithiothreitol, and 10% (v/v) glycerol. The column was developed with a 600-ml linear gradient of 0 to 0.5 M KC1 in the same buffer. A typical elution profile is shown in Fig. 2.

Step 5: Second ammonium sulfate fractionation. To the pooled DEAE-cellulose fractions containing hexokinase activity, solid ammonium sulfate was added to 45% saturation. The suspension was stirred for 30 min and centrifuged at 16,000g for a further 30 min. The ammonium sulfate precipitate was resuspended in 2 to 3 ml of 2.5 mM sodium-potassium phosphate buffer, pH 7.5, and then dialyzed for 3 h against 500 vol of 2.5 mM sodium-potassium phosphate buffer, pH 7.5, containing 10% (v/v) glycerol, with three changes of buffer.

Step 6: A$inity chromatography. The enzyme solution from Step 5 (free of glucose) was applied to a Sepharose-N-aminohex- anoylglucosamine column (1 X 4 cm) equil-

ibrated with 2.5 mM sodium-potassium phosphate, pH 7.5, containing 10% (v/v) glycerol. After the sample application, the affinity column was washed with 5 column volumes of buffer phosphate and then by 2.5 InM sodium-potassium phosphate buffer, pH 7.5, containing 10% (v/v) glycerol and 0.2 M KC1 until the protein absorbance at 280 nm in the eluate was zero. The hexokinase was eluted by 2.5 mM sodium-potassium phosphate buffer, pH 7.5, containing 10% (v/v) glycerol, 0.1 M KCl, and 25 mM glucose (Fig. 3). The preparation obtained at this stage was 290,000-fold purified with a specific activity of 190 U/mg of protein. Hexokinase type III was isolated in a 60% yield as a protein that is homogeneous by sodium dodecyl sulfate-gel electrophoresis. The purification procedure is summarized in Table I.

Preparation of Pig Erythrocyte Hexokinase Type II

Pig red cell hexokinase type II was prepared partially purified in order to study

STOCCHI ET AL.

/-, -2.0 5

c 0 it %

-l.sw

Y

2 K

-1.0%

2

z w

-0.5 &

%

-w-r 10 20 30 40 50

FRACTION NUMBER

FIG. 3. Affinity chromatography of pig erythrocyte hexokinase type III. The sample from Step 5 was applied to the affinity column (1 X 4 cm) equilibrated in 2.5 rnrd sodium-potassium phosphate buffer, pH 7.6, containing 10% (v/v) glycerol, and operated at 10 to 15 ml/h. Fractions of 2.5 ml were collected. At point A 200 mM KCI was added in the developing buffer and the column was washed until the protein absorbance at 280 nm was 0. At point B elution was initiated by adding 100 mM KC1 and 25 rnrd glucose to the equilibrating buffer; 0, absorbance at 280 nm; 0, hexokinase activity.

some of its molecular properties. In this The key step in the preparation of hexo- case the availability of homogeneous hexokinase II was its separation from hexokinase was limited by the low amount of kinase III. This was achieved by ammo- hexokinase II in pig erythrocytes (l-296 of nium sulfate fractionation. In fact, hexo- the total glucose-phosphorylating activity). kinase type III was completely precipitated

TABLE I

PURIFICATION OF HEXOKMASE TYPE III FROM PIG RED BLOOD CELLS

Step Fraction

Specific Volume Protein Activity activity Yield Purification

(ml) (mg/ml) (units/ml) (units/mg) 6) (-fold)

1 Hemolysate 1500 170 0.112 0.00066 100 -

2 DEAE-Sephadex A-50 3300 1.9 0.0504 0.0264 99 40 eluate

3 Ammonium sulfate precipitation, 30 to 45%

22.5 50 7.5 0.15 99 227

4

5

DE 52-cellulose eluate

Ammonium sulfate precipitation, 0 to 45%

170 1.22 0.988 0.820 99 1242

4.2 48 40 0.833 99 1262

6 Immobilised N-acetylglucosamine eluate

14.5 0.036 7 191.5 60 290,000


by ammonium sulfate at 30 to 45% saturation whereas hexokinase type II was precipitated by 60 to 80% ammonium sulfate saturation. Figures 1D and E show that each enzyme fraction was not con- taminated by the other. Similar results were also previously obtained by gel electrophoresis (8). Hexokinase type II from the ammonium sulfate fraction was further purified by DEAE-cellulose chromatography (Whatman DE-52). The purification procedure is summarized in Ta- ble II.

Molecular Weight

The molecular weights of hexokinase types II and III were estimated by gel filtration on Ultrogel AcA44 for three different preparations each and were found to be about 100,000 (Fig. 4). The molecular weight of hexokinase type III was also estimated under denaturing conditions by sodium dodecyl sulfate-gel electrophoresis to be 100,000, indicating that the enzyme is a monomer (Fig. 5). In each case, a least- squares program was used to determine the straight line with a correlation coefficient of about 0.999.

pH Optimum

Hexokinase type II and type III show different pH dependencies of enzyme ac-

.I 4.6 48 5.0 5.2 5.4

Log molecular weight

FIG. 4. Molecular weight estimation of hexokinases II and III by gel filtration on Ultrogel AcA44. The column was calibrated with the protein standards (1) 3-phosphoglycerate kinase; (2) phosphoglucomutase, (3) lactate dehydrogenase; and (4) aldolase.

tivity. Hexokinase type II shows an optimum at pH 9.0 whereas hexokinase type III is most active at pH 8.0 (not shown).

Isoelectric Focusing

Isoelectric focusing of the purified hexokinases under native conditions in glycerol gradient solutions provide single peaks with ~14.3 to 4.4 for hexokinase type III

TABLE II

PREPARATIONOFHEXOKINASETYPE II FROM PIG REDBLOODCELLS

Step Fraction

Specific Volume Protein Activity activity Yield Purification

(ml) bdml) (units/ml) (units/mg) (%I (-fold)

1 Hemolysate 1500 170 0.00112 O.OOOOO65” 100 -

2 DEAE-Sephadex A- 3300 1.9 0.000509 0.000267” 99 41.2 50 eluate

3 Ammonium sulphate precipitation, 60 to 80%

24 40 0.070 0.00175 99 269

4 DE 52-cellulose eluate

22 0.3 0.076 0.25 99 39,160

“The value of the specific activity was calculated after taking into consideration the amount of hexokinase II obtained after chromatographic separation on a DE-52 column.

372 STOCCHI ET AL.

+ tracking dye

FIG. 5. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis of hexokinase type III. Left, calibration curve for the determination of the molecular weight by sodium dodecyl sulfate- gel electrophoresis. Standard proteins and their subunit molecular weights are (1) tripsinogen, PMSF treated (A& = 24,000); (2) pepsin (AZ, = 34,700); (3) egg albumin (J4, = 45,000); (4) bovine serum albumin (A& = 66,000); (5) phosphorylase a (M, = 100,000); (6) #I-galactosidase (M, = 130,000).

and pI 4.75 to 4.85 for hexokinase type II. The same isoelectric points were also found if both hexokinase type II and III were coelectrofocused under the same conditions (Fig. 6).

Kinetic Properties and Spec$city

Hexokinase type II and type III are both able to phosphorylate several hexoses and to utilize different nucleotide triphosphates as phosphate donors. However, hexokinase II shows an apparent K, for glucose of 1.5 * 10e4 M with a negative cooperative effect (nn = 0.4) (Fig. 7) but hexokinase III shows an apparent K,,, for glucose of 1.5. lop5 M and a positive cooperative effect (nn = 1.5). Furthermore, glucose at concentrations higher than 0.4 mM becomes an inhibitor of the enzyme (Fig. 8). In contrast, the apparent K, values for MgATP for both hexokinases are similar and MgGTP, MgUTP, and MgCTP did not serve as phosphate donors at 10 mM concentra-

tions (Table III). Among the hexoses tested, 2-deoxy-D-glucose and mannose are as ef- ficient as glucose for hexokinase type III. All determinations were performed in 0.135 M glycylglycine, pH 8.1, at 30°C.

Temperature Dependence of Hexokinase Type II and Type III

The influence of temperature on the hexokinase activities was studied in the presence of 0.25 InM glucose for hexokinase type III and 100 mM glucose for hexokinase type II (saturating glucose concentrations) and 5 mM MgATP, 5 mM excess Mg in 0.135 M glycylglycine, pH 8.1, for both hexokinases. The results are plotted in Arrhenius plots (log Vagainst l/OK) (Fig. 9).

A breakpoint in the Arrhenius plot was observed at 32°C for hexokinase type III but not for hexokinase type II. The calculated activation energy of hexokinase II is 11.4 kcal/mol and that of hexokinase III is 21.6 kcal/mol at temperatures lower than

PIG RED BLOOD CELL HEXOKINASE

FRACTION NUMBER

FIG. 6. Isoelectric focusing of purified hexokinases II and III. A 0.2-ml sample with 3 units of hexokinase type III and 1 ml of hexokinase type II with 0.2 units was applied to LKB 8100 column in a pH gradient 4 to 8. When the experiment was performed with hexokinase II, only the right peak was obtained but, when hexokinase III was applied, the left peak appeared. 0, Hexokinase activity; * . e, pH gradient.

32°C and 5.7 kcal/mol at higher temperatures.

Enzyme Stability

Pig red blood cell hexokinase type III is very unstable when compared to pig hexo-

r A

[GLUCOBE].IO-3 M

373

kinase type II and hexokinase type I from other sources (4-6). In fact, while hexokinase III, obtained from Step 3 or 4, could be stored for several days at -20°C without loss of enzyme, the affinity chromatography must be performed immediately after Step 5.

0.6 -

0.4 -

> :0.2-

I >o- 1

-0.2-

-0.4.

-0.6 -

111: 1 2 3

Log [GLUCOSE].~M

FIG. 7. Saturation function for glucose of pig erythrocyte hexokinase type II. The enzyme was prepared as described in the text and was used after purification on DEAE-cellulose. This experiment was performed as described under Experimental Procedures. For each value obtained at a glucose concentration higher than 1 mM a blank obtained without MgATP was routinely subtracted. The maximal velocity (17.2 munits) is achieved in presence of 100 mad glucose and is indicated by the horizontal dashed line (A). The nn and Km values were 0.4 and 0.147 mM, respectively, and were obtained from the Hill plot shown in (B).

STOCCHI ET AL.

0.5

0.1

0.1 1

20 40 60 [GLUCOSE]-10-6 M

1 2 3

[GLUCOSE].W~ M

1.0. B

0.6 -

0.6-

0.4 -

.> > 1

0.2-

> o-

B -O.Z-

-0.4.

-0.6.

-O.S-

l.O-

1.2.

0.5 1.0 1.5

Log [GLUCOSE].,A

FIG. 8. Effect of glucose concentrations on hexokinase type III. The highly purified enzyme was obtained as described in the text. The assay mixture was as described under Experimental Procedures. The inset shows the saturation function for glucose at concentrations ranging from 0 to ‘70 PM. The maximal velocity (0.54 units) was achieved in presence of 0.3 mM glucose and 5 mM MgATP as shown in (A). The nu and K,,, values were 1.45 and 0.0144 mrd, respectively, and were obtained from the Hill plot shown in (B). The experiments were conducted at 30°C.

Both hexokinases require sulfhydryl- of glycerol (10 to 15% v/v). The two dif- protecting agents such as dithiothreitol or ferent forms of pig erythrocyte hexokinase 2-mercaptoethanol for optimal stability. were inactivated as a function of time when Both purified hexokinases can be also sta- incubated at 44°C in 5 mM sodium phos- bilized by hexoses such as glucose, fructose, phate buffer, pH 7.5, containing 0.25 mM and mannose, and by high concentrations glucose, 10% (v/v) glycerol, and 1 mM di-

TABLE III

SPECIFICITY OF PIG ER~THROCYTE HEXOKINASES II AND III

Compounds KU”

Hexokinase II

Relative velocity* K,”

Hexokinase III

Relative velocity*

MgATP MgGTP MgITP MgUTP MgCTP @+)-Glucose D(+)-Mannose D(-)-Fructose 2-Deoxy-D-glucose D(+)-Giucosamine N-AcetyI-D-ghicosamine DC+)-Galactose

0.3 100

- - - -

0.147 100 N.D.” N.D.” N.D.” N.D. N.D. N.D. N.D.” N.D.

0.5 100 - -

2.5 24 - -

0.0144 0.014 1.5 0.033 0.2

- -

- -

100 85 12.6

151 35 - -

“Apparent K, values were determined as described in the text. “Maximum velocities are expressed relative to the V,,,, for glucose (100%) of MgATP (100%) when the

enzyme is saturated by the considered substrate. c N.D., not determined.

PIG RED BLOOD CELL HEXOKINASE

3.1 3.2 3.3 34 3.5 ! I/T.103(‘K-‘)

FIG. 9. Arrhenius plot, log Vversus l/T. Incubations were conducted at the temperatures indicated. Incubation time was 26 min. The transition temperature for hexokinase type III is 32°C. The energy of activation was calculated above and below the transition temperature, aa illustrated. (a) Hexokinase type III; (b) hexokinase type II.

thiothreitol. Under these conditions hexokinase type III was more stable than hexokinase type II (not shown).

Amino Acid Composition

The amino acid composition of the purified hexokinase type III is presented in Table IV. It was calculated from three analyses assuming a molecular weight of 100,000.

DISCUSSION

Glucose phosphorylation in mammalian red blood cells is usually catalyzed by hexokinase I or its subtypes (4-7, 19-23). However, the hexokinase isozymic pattern of pig erythrocytes shows the presence of two distinct glucose-phosphorylating activities, identified by chromatographic and kinetic properties as hexokinase type II and III.

Hexokinase III is the predominant molecular form in adult pig erythrocytes, ac- counting for 98-99s of the total glucose- phosphorylating activity. Newborn pig erythrocytes, in contrast, contain about one-third of the total hexokinase activity found in adult pig red blood cells and pos- sess hexokinase type II as the major form. In similar studies with pig erythrocytes previously reported from this laboratory (S), because of the negative cooperativity of hexokinase type II, this was erroneously

classified as “similar to hepatic glucokinase” (8). Dixon and Wilson (9) have also been able to provide evidence for two distinct isozymes of hexokinase in adult pig erythrocytes; however, these isozymes were

TABLE IV

AMINO ACID ANALYSIS OF PIG ERYTHROCYTE HEXOKINASE TYPE III

Amino acid Residues”

Aspartic acid 49 Threonine* 32 Serine* 110 Glutamic acid 121 Glycine 212 Alanine 136 Cysteine Trace Valine 54 Methionine 10 Isoleucine 31 Leucine 60 Tyrosine 5 Phenylalanine 17 Histidine 14 Lysine 19 Arginine 19 Tryptophan” 2

a The number of amino acid residues was calculated on the basis of a molecular weight of 109,096.

*The values for serine and threonine were not cor- rected for destruction during acid hydrolysis.

’ Tryptophan was determined by the method of Bliss (16).

376 STOCCHI ET AL.

both classified as two distinct type III hexokinases. The evidence provided in this paper provides strong support that the two pig erythrocytes’ glucose-phosphorylating activities correspond to hexokinase types II and III.

The purification developed for pig erythrocytes hexokinase III reported here is a reliable and fast technique. This procedure has been performed at least 10 times with the same results. A partial purification of pig erythrocyte hexokinase has previously been described by other investigators (9) but with a final specific activity of 0.313 units/mg of protein compared with the value of 190 units/mg protein in the present report, similar to that obtained for homogeneous hexokinase III from rat liver (24).

The molecular weight of the pure pig erythrocyte hexokinase III found in our experiments is similar to mammalian hexokinase types I, II, and III (4-6,10,18, 24). Hexokinase type III possesses the highest affinity for glucose (Km = 1.4. 10m5 M) and shows a positive cooperative effect (nn = 1.5). Of interest, a similar positive cooperative effect for a monomeric enzyme has also been reported for glucokinase (26) and a “mnemonical” mechanism has been proposed to explain this phenomenon. This is a highly unusual property for a monomeric enzyme that, in the case of hexokinase type III, is also associated with the inhibition exerted by the same substrate.

REFERENCES

1. PURICH, D. L., FROMM, H. J., AND RUDOLPH, F. B. (1973) Advan EnzymoL 39,249-326.

2. COLOWICK, S. P. (1973) in The Enzymes (Boyer, P. D., ed.), Vol. 9, pp. l-48, Academic Press, New York.

3. WALKER, D. G. (1966) in Essay in Biochemistry (Campbell, P. M., and Greville, G. D., eds.), Vol. 2, pp. 36-67, Academic Press, New York.

4. MAGNANI, M., DACHA, M., STOCCHI, V., NINFALI, P., AND FORNAINI, G. (1980) J. Biol Chem 255, 1752-1756.

5. STOCCHI, V., MAGNANI, M., CANESTRARI, F., DACHA, M., AND FORNAINI, G. (1981) J. Biol Chem 256, 7856-7862.

6. STOCCHI, V., MAGNANI, M., CANESTRARI, F., DACH& M., AND FORNAINI, G. (1982) J. Biol Chem. 257, 2357-2364.

7. MAGNANI. M., STOCCHI, V., CANESTRARI, F., DACHA. M., AAD FORNAINI, G. (1982) Biochem. Int. 4, 673-677.

8.

9.

10.

11.

12.

13.

14. 15.

16. 17.

18.

19.

20.

FORNAINI, G., DACHA, M., MAGNANI, M., FAZI, A., CUPPINI, C., AND Bossir, M. (1978) Enzyme 23, 46-51.

DIXON, E., AND WILSON, B. A. (1981) J. Exp. ZooL 215,63-76.

KATZEN, H. M., AND SCHIMKE, R. T. (1965) Proc. Natl. Acad Sci USA 54,1218-1225.

MAGNANI, M., STOCCHI, V., SERAFINI, N., PIATTI, E., DACHA, M., AND FORNAINI, G. (1983) Arch Biochem Biophys. 226.377-387.

BEUTLER, E. (1975) Red Cell Metabolism, 2nd ed., p. 11, Grune & Stratton, New York.

LOWRY, 0. K., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Bi01 Chem 193, 265-275.

LAEMLI, U. K. (1970) Nature (London) 227, 680. WEBER, K., AND OSBORN, M. (1969) J. Biol Chem

244,4406-4412. BLISS, R. D. (1979) Anal Biochem 93, 390-398. GROSSBARD, L., AND SCHIMKE, T. R. (1966) .I. Bid

Chem 241,3546-3560. URETA, T. (1975) in Isozymes (Markert, C. L., ed.),

Vol. 3, pp. 575-601, Academic Press, New York. RIJKSEN, G., AND STAAL, G. E. J. (1977) B&him

Biophys. Acta 435, 75-86. RIJKSEN, G., JANSEN, G., KRAAIJENHAGEN, R. G.,

VAN DER VLIST, M. J. M., VLUG, A. M. C., AND STAAL, G. E. J. (1981) Biochim Biophys. Acta 659,292-301.

21. GARH, M. (1980) Hqppe-Se&r’s Z. Physiol Chem 361,829~837.

22. GAHR, M. (1980) Brit. J. HaemutoL 46, 529-535. 23. GERBER, G., PREISSLER, H., HEINRICH, R., AND

RAPOPORT, S. M. (1974) Eur. J. Biochem 45, 39-52.

24. WRIGHT, C. L., WARSY, A. S., HOLROYDE, M. J., AND TRAYER, J. P. (1978) Biochem J. 175.122- 135.

25. MAGNANI, M., SERAFINI, G., STOCCHI, V., Bossb, M., AND DACHA, M. (1982) Arch Biochem Bio- phys. 216, 449-454.

26. GREGORIOU, M., TRAYER, I. P., AND CORNISH-BOW- DEN. A. (1981) Biochemistru 20.499-506.

Pig red blood cell hexokinase: Evidence for the presence of hexokinase types II and III, and their purification and characterization

Documents